Chemical analysis of liquids, such as water, milk and biological fluids is often desirable or necessary for health maintenance and diagnosis and treatment of disease. Various compositions and elements to facilitate such analyses are known. Such materials generally comprise a reagent composition for determining the substance under analysis, identified herein as an analyte. The analyte can be living cells, such as yeast cells, white blood cells, bacteria or other living organisms, or a nonliving chemical substance such as an enzyme. The reagent composition, upon interaction with the analyte, provides a detectable change (for example, dye formation) which can be quantified in some manner.
The determination of specific hydrolytic enzymes in biological fluids can be useful for the diagnosis and treatment of diseases. It can also be useful for determining the presence of certain microorganisms because the metabolism of the microorganism is dependent upon the presence of certain hydrolytic enzymes.
A number of analytical procedures have been developed whereby a substrate for an enzyme of interest is hydrolyzed to release a detectable moiety. These procedures use both colorimetric and fluorometric dyes. Fluorometric assays are generally preferred because of generally greater sensitivity. E. P. Publication 122,148 describes an assay for microorganisms using certain coumarin derivatives as substrates which release dyes when the substrate is hydrolyzed. Other known assays utilize diazonium compounds to provide detectable moieties in response to hydrolytic analytes (see U.K. Patent Application 2,031,949).
A significant advance in the art is described in U.S. Ser. No. 824,766, filed Jan. 31, 1986, entitled REDUCIBLE COMPOUNDS AND ANALYTICAL COMPOSITIONS, ELEMENTS AND METHODS OF UTILIZING SAME. This application describes useful reducible compounds which, when reduced by a reductant (such as NADH) in a test sample, provide a detectable species, such as a chromogen or fluorogen. Many of the detectable species so released have high extinction coefficients, thus providing high sensitivity to low concentrations of analytes. It would be desirable to use such compounds in the detection of hydrolytic analytes, such as hydrolytic enzymes found in various organisms. However, hydrolytic enzymes and their substrates are not always reductants for such reducible compounds.
It has been found that the reduction of some reducible compounds can be undesirably slow. In such cases, an electron transfer agent can be used to facilitate compound reduction. However, even in these reactions, only one equivalent of detectable species is released for each equivalent of electron transfer agent used. Therefore, it would be desirable to have a more sensitive assay, that is, an assay wherein more than one equivalent of detectable species is released from only one equivalent of electron transfer agent.